The nematicidal potential of novel fungus, Trichoderma asperellum FbMi6 against Meloidogyne incognita

One of the most damaging pests in vegetable crops is the root-knot nematode (Meloidogyne incognita) worldwide. The continuous use of nematicide is costly and has unintended consequences for human and environmental health. To minimize nematicides, eco-friendly integrated nematode management is required. Trichoderma, an antagonistic fungus has been explored to control root-knot nematode. The fungal bio-control strain FbMi6 was identified as Trichoderma asperellum (accession no. MT529846.1). T. asperellum FbMi6 showed substantial nematicidal activity in the laboratory, with egg hatch suppression (96.6%) and juvenile mortality (90.3%) of M. incognita. T. asperellum FbMi6 was examined under pot and field conditions (after neem cake enrichment), both alone and in combination, and compared with controls. Application of T. asperellum FbMi6 enriched neem cake (1-ton ha-1) increased (28.3%) the okra yield and decreased (57.1%) nematode population as compared with control. T. asperellum FbMi6 enriched neem cake had higher polyphenol content (resistance enhancer) in okra compared with inoculated check.

). Based on morphological and molecular characteristics, isolate FbMi6 was identified as T. asperellum. The sequence was submitted to GenBank (NCBI), with accession number MT529846.1. It showed 100% similarity to T. asperellum as per GenBank data. Also, according to phylogenetic analysis, FbMi6 was found most closely related to T. asperellum (Fig. 2). In comparison with the control, T. asperellum FbMi6 significantly reduced hatchability and having maximal juvenile mortality. T. asperellum FbMi6 inhibited egg hatching in all concentrations as compared to control. At 120 h, maximum egg hatching inhibition (96.6%) was observed at 80% concentration of T. asperellum FbMi6 as compared with control. With increasing exposure period, egg hatching inhibition gradually increased. T. asperellum FbMi6 culture filtrates cause egg deformation and hatching suppression starting at 24 h, in all the concentrations (Fig. 3).
Similarly, T. asperellum FbMi6 culture filtrate showed antagonistic or nematocidal activity against M. incognita juveniles in all the concentrations (Fig. 4). T. asperellum FbMi6 showed maximum root-knot nematode juveniles' mortality in all concentrations at 72 h exposure as compared to control. T. asperellum FbMi6 at 80% concentration had the highest (90.3%) juvenile mortality compared to control. The mortality of juveniles enhanced as exposure time increased (Fig. 4).

Effects of T. asperellum
FbMi6 enriched neem cake on M. incognita and vegetative growth of okra. The application of T. asperellum FbMi6 enriched neem cake had substantial (P ≤ 0.05) suppressive effects on M. incognita population in terms of J 2 s 200 -1 cc soil, galls and egg masses plant −1 , and eggs per egg mass, as well as promote the vegetative growth of okra. The gall index and egg masses were significantly reduced with T. asperellum FbMi6 enriched neem cake (Table 1). Fewer galls on okra roots were observed with T. asperellum FbMi6 enriched neem cake at 20 g kg −1 soil. Egg masses and eggs per egg mass were also lower in all T. asperellum FbMi6 enriched neem cake treatments compared with control. In comparison to the untreated inoculated check, soil nematode population was considerably reduced in all the treatments of T. asperellum FbMi6 enriched neem cake. In contrast to the untreated control, T. asperellum FbMi6 enriched neem cake had the lowest nematode population at 20 g kg −1 soil application.
Plant biomass (shoot and root weight) differed significantly between T. asperellum FbMi6 enriched neem cake and control (untreated). When compared to the untreated inoculated control, all treatments with T. asperellum FbMi6 enriched neem cake significantly enhanced plant height (P ≤ 0.05). The results ( Table 2) showed that T. asperellum FbMi6 enriched neem cake at 20 g kg −1 soil had considerably higher plant height than T. asperellum    T. asperellum at 20 % conc.

Discussion
Many countries, including the United States, Australia, India, and China, have recently added to the collection of Trichoderma sources. Trichoderma research first concentrated on soil flora and fauna, soil remediation, soil biology, pollution, and general mycology. As our understanding of Trichoderma grew, researchers concentrated on biocontrol and its applications in plant nematology and mycology. Isolate FbMi6 was identified as T. asperellum based on cultural, morphological, and molecular findings in this study. Trichoderma species has also been found in diversified environment and isolated from the upper atmosphere 24 , wetlands 25 , and rhizosphere soil 26 . There have been 141 or more Trichoderma species recorded worldwide [27][28][29][30] . T. asperellum FbMi6 showed substantial antagonistic activity on egg hatching and juvenile mortality of M. incognita in present investigation, suggesting a possible nematoxic compounds in it. Many scientists have already been conducted research on Trichoderma isolates for nematode inhibition in vitro [31][32][33][34] . Deformation of eggs and juveniles confirmed the presence of nematicidal compounds in culture filtrates of Trichoderma. T. asperellum FbMi6 generated nematicidal chemicals that appeared to play a key role in nematode death. Plant parasitic nematode infestations have been reduced using Trichoderma species as biocontrol agents 35,36 . Biocontrol effectiveness of these microbes was demonstrated by the antagonism of Trichoderma culture filtrates against M. incognita. Meyer 37 observed that 253 fungal isolates (Acremonium sp., Aspergillus sp., Fusarium sp., Paecilomyces sp.) had a nematicidal influence on juvenile development and egg hatch suppression. The exceptional performance of Trichoderma enriched neem cake under pot and field circumstances is a glimmer in the nematology picture in the current study. The current findings were consistent with those of Affokpon 38 , who found that T. asperellum suppressed root-knot nematode and increased plant tolerance. Trichoderma asperellum biocontrol activity is attributed to the buildup of phytoalexins 39 . Increased plant growth of okra with Trichoderma enriched neem cake treated plants due to the reduction of root-knot nematode population. www.nature.com/scientificreports/ In comparison to untreated plants, treated plant roots absorb more nutrients from the soil. Several studies have shown that combining biocontrol agents with organic amendments increases plant growth and yield while also suppressing nematode populations [40][41][42] . However, there is a scarcity of information on the utilization of T. asperellum as a biocontrol agent against M. incognita. Present study showed that T. asperellum enriched neem cake can be used to control the root-knot nematode in vegetable crops. Nematode populations were found to be reduced in Trichoderma enriched neem cake compared to control. Plant parasitic nematodes are considerably reduced by neem and its derivatives 43,44 . The presence of toxic chemicals such as azadirachtin and nimbin (secondary metabolites) present in all regions of neem, which have a protective role against nematode infection and inhibited nematode proliferation 45 .
T. asperellum FbMi6 enriched neem cake improves plant tolerance in nematode-infected plants by boosting biochemical and physiological attributes such as polyphenols, total chlorophyll, nitrogen balance index, anthocyanin, and flavonoids as compared to control. The formation of secondary metabolites such as phenolic compounds hindered nematode reproduction 46 . Flavonoid inhibits nematode movement and egg hatching 47 , and plays an important function in plant defense 48 . Phenolic chemicals are produced in response to nematode stress in plants 49,50 . The amount of phenolic content accumulated in a plant determines its level of stress tolerance 51 . In comparison to the untreated inoculated control, use of bio-agents (Pseudomonas aeruginosa) in combination with neem cake produces systemic resistance in cotton 52 . Ammonia and higher the C/N ratio of organic inputs showed nematicidal action against plant parasitic nematodes. These substances may also affect root-knot nematode egg viability and hatching 53,54 . Organic additions alter the physiochemical and physical properties of the soil, which has a deleterious effect on nematode motility and host finding 55 . The similar technique may have worked against the root-knot nematode in okra. Trichoderma is a well-known, worldwide recognized biocontrol agent. Nonetheless, studies on Trichoderma spp. have focused on one or more parameters in a single strain. T. asperellum, the identified strain, has a quicker growth rate and a substantial nematocidal effects on M. incognita. According to our findings, pot and field studies showed that T. asperellum FbMi6 enriched neem cake considerably reduced the population of M. incognita. The remarkable effectiveness of T. asperellum FbMi6 enriched neem cake against root-knot nematode in outdoor circumstances was proved.

Materials and methods
Isolation and identification of bioagent. All methods were carried out in accordance with relevant guidelines and regulations. Wet soil samples were obtained from various places in Haryana, India. The location does not require any special permission. Trichoderma isolates were isolated on potato dextrose agar (PDA) culture plates with 50 μg mL −1 streptomycin from collected samples using the serial dilution method 56 and culture was multiplied on potato dextrose broth (PDB) at 25 ± 2 °C in a BOD incubator.
The Trichoderma isolate was identified at the generic level based on morphological parameters such as growth pattern and colony colour. Genomic DNA was isolated in pure form from the culture plates. The ITS-rDNA partial gene was effectively amplified using primers ITS4 and ITS5. The ABI-BigDye ® Terminatorv3.1 Cycle Sequencing Kit was used to set up the sequencing PCR. Sequence generated was manually modified for uniformity using the ABI 3100 automated DNA sequencers. Further To determine identification, the sequence data was aligned with publicly accessible sequences and compared with the GenBank database using BLSATN. The sequences were aligned in ClustalX. The partial ITS-rDNA sequence was submitted to GenBank (NCBI) to get the accession number. MegaX software was used to construct a phylogenetic tree of related Trichoderma species through the maximum likelihood method 57 . This culture was given the name FbMi6 and sent to the National Fungal Culture Collection (NFCCI) in Pune, Maharashtra, India for storage (http:// nfcci. aripu ne. org/). Obtaining M. incognita eggs and second-stage juveniles. Meloidogyne incognita eggs and J 2 s were collected from a pure population kept on brinjal (Solanum melongena L.) cv. Hisar Shyamal at CCSHAU, Hisar, Haryana, India. The eggs were collected from infested brinjal roots using the sodium hypochlorite method 58 . The juveniles were extracted using cobb's method 59 followed by Modified Baermann Funnel Technique (MBFT) 60 .

Maintenance of bio-agents. Trichoderma asperellum
In vitro assay. Culture broth 100 mL was centrifuged for 20 min at 1500 rpm containing 1 × 10 8 spores (CFU mL −1 ). Cell-free culture filtrates were recovered through 0.45 μm filters (Whatman™). Culture filtrate was checked for the presence of any fungal spores using PDA plating procedures. For Egg hatching inhibition assay, five surface sterilized egg masses were kept into tissue culture plates with four different concentrations at 20, 40, 60 and 80% of T. asperellum and distilled water and PDB were kept as control. The hatched juveniles were counted under a stereoscopic binocular microscope for alternate day upto 10 days and the percent hatching inhibition computed.
Juveniles' mortality assay was conducted in tissue culture plates with five mL culture filtrates at four different concentrations (20,40,60 and 80%) with water and PDB as control. Hundred newly hatched J 2 were transferred in each tissue culture plate well. Death of juveniles was examined at 24 h intervals up to 72 h under a stereo zoom binocular microscope (Gippon). The death of juveniles was confirmed after recovering in fresh water. All hatching and mortality assay were repeated twice and assay were conducted at room temperature (25 ± 2 °C). www.nature.com/scientificreports/ Screen house assay. One ton of neem cake (organic carbon-10.45%, nitrogen-0.84%, phosphorous-0.69%, Potassium-0.59%) was enriched with three liters of T. asperellum FbMi6 aqueous solution. Mixed biomass (T. asperellum and neem cake) was kept under shade for 15 days with a moisture content of 25-30% and temperature of 25-28 °C for proper enrichment. T. asperellum CFU were measured in neem cake after enrichment using a serial dilution procedure (56). Before sowing, experimental pots were filled with T. asperellum FbMi6 enriched neem cake. The experiment was conducted in earthen pots (15 cm diameter) in a screen house at CCSHAU Hisar, India (29°10' N; 75°46' E). Treatments as: T. asperellum FbMi6 enriched neem cake (5 g kg −1 soil), T. asperellum FbMi6 enriched neem cake (10 g kg −1 soil), T. asperellum FbMi6 enriched neem cake (15 g kg −1 soil), T. asperellum FbMi6 enriched neem cake (20 g kg −1 soil), Carbofuran 3G (16 mg kg −1 soil) were applied respectively. Five replications of each treatment were used in a completely randomized design (CRD). Okra cv. Hisar unnat seeds were sowed, and one plant per pot was maintained after thinning. Freshly hatched 2000 J 2 s were inoculated in steam sterilized soil at sowing time. Hoagland solution was applied to plants 61 .
Plant height, fresh shoot and root weight, dried shoot and root weight of okra, nematode galls per plant, egg masses per plant, eggs per egg mass, second stage juveniles per 200 cc soil were measured at harvesting (45 days after sowing). Galls and egg masses were counted with the help of a hand lens and soil was processed by Cobb sieving method 59 .
After drying in a hot air oven, physiological and biochemical characteristics of okra were recorded. Folin-Ciocalteau assay was used to determine total phenolic content 62 . Oven dried 0.5 g samples were extracted in 10 ml ethanol (80%) and centrifuged at 10,000 rpm for 20 min. The supernatant was collected and evaporated until completely dry. Reagent FC (1 N) 100 µl and 20 µl of each extract were placed in test tubes and left for 8 min before adding 300 µl of sodium carbonate. For 30 min at 40 °C, the contents were allowed to incubate in the dark. At 765 nm, the absorbance was measured. On a dry weight basis, the phenolic content of the sample was reported in mg GAE/100 g of sample. Dualex sensor (Dualex ® Scientific sensor) was used to calculate total chlorophyll, NBI, anthocyanin, and flavonoid.
Neem cake 1 ton ha −1 , T. asperellum FbMi 6 enriched neem cake 1 ton ha −1 , chemical check (33 kg Furadan 3G ha −1 ), and inoculated control were used in a Randomized Block Design (RBD) with five replications. Observations on root gall index and final nematode population were recorded at 90 days after seeding. Cobb's methods were used to assess the soil population 59 . Okra yield and root gall index 63 were also measured. Statistical analysis. SPSS version 10.0 was used to analyze the data statistically. The means were separated using DMRT. Differences in treatment means were considered significant at P ≤ 0.05. Values with the different letter in a column are significantly different at P ≤ 0.05. SD was calculated from excel spreadsheet Ver. 16. Ethics approval and consent. In conducting this study, experiments on live vertebrates and/or higher invertebrates were not required any special permission for approval and consent. The variety of okra Hisar Unnat was developed by CCSHAU, Hisar therefore, it not required any permission. All images used in the manuscript are original.

Data availability
All data generated or analyzed during this study are included in this published article (and its Supplementary Information files). The sequence data obtained in this study are openly available in GenBank of NCBI at https:// www. ncbi. nlm. nih. gov/ under the Accession No. MT529837.1.